In eukaryotic cells DNA is packaged with histones to form chromatin. Approximately 150 base pairs of DNA are wrapped twice around an octamer of histones (two each of histones 2A, 2B, 3 and 4) to form a nucleosome, the basic unit of chromatin. Changes in the ordered structure of chromatin can lead to alterations in transcription of associated genes. This process is highly controlled because changes in gene expression patterns can profoundly affect fundamental cellular processes, such as differentiation, proliferation and apoptosis. Control of changes in chromatin structure (and hence of transcription) is mediated by covalent modifications to histones, most notably of their N-terminal tails. These modifications are often referred to as epigenetic because they can lead to heritable changes in gene expression, but they do not affect the sequence of the DNA itself. Covalent modifications (for example, methylation, acetylation, phosphorylation and ubiquitination) of the side chains of amino acids are enzymatically mediated.
The selective addition of methyl groups to specific amino acid sites on histones is controlled by the action of a unique family of enzymes known as histone methyltransferases (HMTs). The level of expression of a particular gene is influenced by the presence or absence of one or more methyl groups at a relevant histone site. The specific effect of a methyl group at a particular histone site persists until the methyl group is removed by a histone demethylase, or until the modified histone is replaced through nucleosome turnover. In a like manner, other enzyme classes can decorate DNA and histones with other chemical species, and still other enzymes can remove these species to provide control of gene expression.
The orchestrated collection of biochemical systems behind transcriptional regulation must be tightly controlled in order for cell growth and differentiation to proceed optimally. Disease states result when these controls are disrupted by aberrant expression and/or activity of the enzymes responsible for DNA and histone modification. In human cancers, for example, there is a growing body of evidence to suggest that dysregulated epigenetic enzyme activity contributes to the uncontrolled cell proliferation associated with cancer as well as other cancer-relevant phenotypes such as enhanced cell migration and invasion. Beyond cancer, there is growing evidence for a role of epigenetic enzymes in a number of other human diseases, including metabolic diseases (such as diabetes), inflammatory diseases (such as Crohn's disease), neurodegenerative diseases (such as Alzheimer's disease), and cardiovascular diseases. Therefore, selectively modulating the aberrant action of epigenetic enzymes holds great promise for the treatment of a range of diseases.
Enhancer of Zeste Homolog 2 (Drosophila) (EZH2) catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3), with its prominent function being to adjust the structure of chromosome. A variety of tumors have high expression of EZH2, which is closely related to the malignant process, invasiveness and metastasis of tumors. Main functions of EZH2 comprise catalyzing methylation of histone, participating in DNA methylation and interfering with DNA repair. EZH2 is a member of the PcG (polycomb-group) gene family. Two complexes, PRC1 (polycomb repressive complex 1) and PRC2 (polycomb repressive complex 2), respectively play a role in maintaining gene suppression and initialing gene silencing. EZH2 gene, together with EED and SUZ12 constitute PRC2 complex, wherein EZH2, as the catalytic subunit of PRC2, can catalyze H3K27m3 and H3K9m3 via its highly conserved SET region in histone methyltransferase, thereby suppressing transcription and regulating gene activity at chromosome level. EZH2 in PRC2 and PRC3 is able to interact with DNA methyltransferase to enhance its activity. Studies have shown that EZH2 is required in binding between some target genes of EZH2 and DNA methyltransferase. In addition, EZH2 is also needed in assisting the methylation of promoters of EZH2-targeted genes. EZH2 plays a role in the recruitment of DNA methyltransferases.
Biochemical and genetic studies have provided evidence that Drosophila PcG proteins function in at least two distinct protein complexes, the Polycomb repressive complex 1 (PRC 1) and the ESC-E(Z) complex (also known as Polycomb repressive complex 2 (PRC2)), although the compositions of the complexes may be dynamic (Otte et al. Curr OpinGenet Dev, 2003, 13:448-54). Studies in Drosophila and mammalian cells have demonstrated that the ESC-E(Z)/EED-EZH2 (i.e., PRC2) complexes have intrinsic histone methyltransferase activity. The complexes generally contain EED, EZH2, SUZ12, and RbAp48 or Drosophila homologs thereof. However, a reconstituted complex comprising only EED, EZH2, and SUZ12 retains histone methyltransferase activity for lysine 27 of histone H3 (U.S. Pat. No. 7,563,589).
Of the various proteins making up PRC2 complexes, EZH2 (Enhancer of Zeste Homolog 2) is the catalytic subunit. The catalytic site of EZH2 in turn is present within a SET domain, a highly conserved sequence motif (named after Su(var)3-9, Enhancer of Zeste, Trithorax) that is found in several chromatin-associated proteins, including members of both the Trithorax group and Polycomb group. SET domain is characteristic of all known histone lysine methyltransferases except the H3-K79 methyltransferase DOT1.
Consistent with a role of EZH2 in maintaining the epigenetic modification patterns of pluripotent epiblast cells, Cre-mediated deletion of EZH2 results in loss of histone H3-K27 methylation in the cells. Further, studies in prostate and breast cancer cell lines and tissues have revealed a strong correlation between the levels of EZH2 and SUZ12 and the invasiveness of these cancers (Bracken et al. (2003) EMBO J 22:5323-35; Kirmizis et al. (2003) Mol Cancer Ther 2:113-21; Kleer et al. (2003) Proc Natl Acad Sci USA 100:11606-11; Varambally et al. (2002) Nature 419:624-9).
Recently, somatic mutations of EZH2 were reported to be associated with follicular lymphoma (FL) and the germinal center B cell-like (GCB) subtype of diffuse large B-cell lymphoma (DLBCL) (Morin et al. (2010) Nat Genet. 42:181-5). In all cases, occurrence of the mutant EZH2 gene was found to be heterozygous, and expression of both wild-type and mutant alleles was detected in the mutant samples profiled by transcriptome sequencing. Currently, the R-CHOP approach has been a standard therapy for most diffuse large B-cell lymphoma (DLBCL).
Small molecule inhibitors of EZH2 that has entered phase II clinical testing so far include EPZ6438 (Tazemetostat) which is used for treating non-Hodgkin B-cell lymphoma (see U.S. Pat. No. 8,765,732B2, US20140128393A1, US20151163A1). In addition, also included is GSK126 (CAS No.: 1346574-57-9) developed by GSK which has currently entered into clinical phase I and which is also a small molecule inhibitor of EZH2 for treating diffuse large B-cell lymphoma and follicular lymphoma.